The objectives of this grant, which has been funded for almost 30 years, are to understand the genetic and epigenetic basis of human bladder cancer. We have defined the existence of two molecular pathways for the genesis of bladder cancer (UC) in which superficial (Ta/T1) tumors, which frequently recur, are distinct from more aggressive tumors at the molecular level. We have also shown profound epigenetic alterations which occur during bladder carcinogenesis and want to continue our studies by using more global approaches to define key genes which may play a role in this prevalent but understudied disease. In the next five year period of the grant, we will use a series of eight hypermethylation markers to complete the examination of DNA in urine sediments obtained from individuals with low grade tumors to determine whether we can detect the frequent recurrences of these tumors. We shall also complete the analysis of DNA from healthy individuals of different ages to determine whether age-related changes in DNA methylation can be detected in urine sediments. Secondly, using high-throughput approaches on the Illumina platform, we have observed a hypomethylation phenotype not seen in the apparently normal urothelium but particularly prevalent in superficial tumors and less frequently in more invasive tumors. This hypomethylation is seen in the vicinity of transcription start sites and might play a role in ectopic gene activation in tumors. In Specific Aims 3 and 4 we will determine the functional significance of these changes by analyzing directly whether methylation of non- CpG island regions which constitute the bulk of the hypomethylation phenotype might be involved in gene activation and have chromatin properties associated with active genes. Finally, in Specific Aim 5 we will take advantage of ongoing clinical trials in which patients with myelodysplastic syndrome are being treated with the hypomethylating drug 5-azacytidine (5-aza-CR). Since we can easily and non-invasively obtain urine sediments from these patients, we can directly test the hypothesis that systemic administration of hypomethylating drugs leads to demethylation of bladder urothelial cells which may have implications in the future for treatment of this disease. PUBLIC HEALTH RELEVANCE: Bladder cancer is the 5th most common cancer in the United States and is one of the most expensive cancers to treat because of its propensity to recur. We have discovered substantial epigenomic changes in low grade (Ta/T1) tumors which can be detected in urine sediments and might help steer clinical decisions associated with gene activation in the tumors, and shall investigate the mechanism for this action. We want eventually to develop epigenetic therapies for bladder cancer and shall take advantage of clinical trials in which patients who do not have this disease are being treated with demethylating agents to test the effects of these drugs on DNA methylation patterns in the normal urothelial cells of humans.